Isolation, expansion and use of clonogenic endothelial progenitor cells

ABSTRACT

A hierarchy of endothelial colony forming cells (EPCs) was identified from mammalian cord blood, umbilical vein and aorta. A newly isolated cell named high proliferative potential-endothelial colony forming cell (HPP-ECFC) was isolated and characterized. Single cell assays were developed that test the proliferative and clonogenic potential of endothelial cells derived from cord blood, or from HUVECs and HAECs. EPCs were found to reside in vessel walls. Use of a feeder layer of cells derived from high proliferative potential-endothelial colony forming cells (HPP-ECPCS) from human umbilical cord blood, stimulates growth and survival of repopulating hematopoietic stem and progenitor cells. Stimulation of growth and survival was determined by increased numbers of progenitor cells in in vitro cultures and increased levels of human cell engraftment in the NOD/SCID immunodeficient mouse transplant system.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/055,182, filed on Feb. 9, 2005, which claims priority from U.S. Ser.No. 60/543,114 filed Feb. 9, 2004, U.S. Ser. No. 60/573,052 filed May24, 2004 and U.S. Ser. No. 60/637,095 filed Dec. 17, 2004, thedisclosures of which are hereby incorporated by reference in theirentireties.

BACKGROUND OF THE INVENTION

Models for stem cell differentiation leading to endothelial andhematopoietic cells are of interest because of the clinical value ofstem cells and their progeny. A hallmark of stem and progenitor cells istheir ability to proliferate and give rise to functional progeny, andprogenitor cells are identified by their clonogenic potential. Methodspreviously used do not guarantee that single endothelial cells have beenisolated and characterized to identify the progenitors.

Endothelial cell proliferation in vivo in normal, mature arterial,venous, and capillary vessels in most mammals is reported to beextremely low, if not nonexistent. In some experimental animals, such aspigs and dogs, radiolabeling studies have demonstrated 0.6-3.0%endothelial cell turnover daily with the dividing cells restricted tofocal areas in certain vessels. Whether these dividing endothelial cellsare unique and possess proliferative potential that is lacking in othermature endothelium remains undetermined.

In marked contrast, plating of endothelial cells derived from human oranimal vessels in vitro is associated initially with brisk endothelialcell proliferation. For example, human umbilical vein endothelial cells(HUVEC) and bovine aortic endothelial cells (BAEC) are two commonlystudied models for in vitro analysis of endothelial cell functions. BothHUVEC and BAEC cells proliferate well initially in culture but celldivision wanes with time and cells become senescent and fail to divideafter 15-20 passages. It is unknown if each endothelial cell derivedfrom the vessels possesses similar proliferative potential or if onlysome of the cells can divide.

Angiogenesis (neoangiogenesis) is the process of new vessel formationfrom pre-existing vessels; this is the process reported to give rise tonew vessels in adult subjects. Recently, bone marrow derived circulatingendothelial progenitor cells (EPCs) have been described and these cellshave also been reported to play a role in new vessel formation, at leastin some experimental murine ischemic or tumor models. Conflictingevidence indicates that bone marrow derived (EPCs) do not contribute tothe endothelial lining of normal arterial, venous, and capillary vesselsduring development and play only a minor role in neoangiogenesis. Arelationship between circulating EPCs and the endothelial cells withproliferative potential that reside in normal vessels is unknown.

Emerging evidence to support the use of EPCs for angiogenic therapies oras biomarkers to assess a patient's cardiovascular disease risk andprogression is accumulating and is generating enthusiasm. However, thereis no uniform definition of an EPC, which makes interpretation of thesestudies problematic and prohibits reproduction of cell types suitablefor clinical use. Although a hallmark of stem and progenitor cells (e.g.hematopoietic, intestinal, neuronal) is their ability to proliferate andgive rise to functional progeny, EPCs are primarily defined by theexpression of selected cell surface antigens. Sole dependence on cellsurface expression of molecules can be problematic because theexpression may vary with the physiologic state of the cell. No assay isreported to assess the proliferative potential (an intrinsic response)in individual endothelial cells or EPCs and thus, no comparativeanalysis is available.

Previous studies reported that populations of cells termed “endothelialprogenitor cells” can be isolated from human umbilical cord blood oradult peripheral blood by culturing either sorted cells expressing thecell surface antigen CD34, or mononuclear cells in defined cultureconditions.

Hematopoietic and endothelial progenitor cells share a number of cellsurface markers in the developing yolk sac and embryo, and geneticdisruption of numerous genes affects both hematopoietic and endothelialcell development. Therefore, these lineages are hypothesized tooriginate from a common precursor, the hemangioblast. A hierarchy ofstem and progenitor cells in hematopoietic cell development is reported.Hematopoietic progenitor cells within the hierarchy are identified bytheir clonogenic and proliferative potential. Although genetic studiesclearly show that the origin of endothelial cells is closely linked tohematopoietic cell development, evidence to support a similar hierarchyof stem and progenitor endothelial cells based on differences inproliferative potential has not been established. That is, a hierarchyof EPCs that can be discriminated by the clonogenic and proliferativepotential of individual cells analogous to the hematopoietic cell systemhas not been reported.

Both hematopoietic stem and progenitor cells (HSC/Ps) are enriched inumbilical cord compared to adult peripheral blood. Cord blood iscurrently used as an alternative resource of hematopoietic stem cellsfor transplantation of patients with a variety of hematologicaldisorders and malignancies.

Thousands of patients require a hematopoietic stem cell (HSC) transplanteach year. Nearly ⅔ of the patients are unable to find a human leukocyteantigen (HLA) compatible match for the transplant. This is particularlytrue for many ethnic populations and under-represented minorities. Only⅓ of Caucasian patients find suitable matched sibling grafts—the mostcompatible source with the least graft versus host disease (GVHD)complications.

Human umbilical cord blood is known to be an alternative source of HSCsfor clinical transplantation. Whether or not the donor cord blood is afull major histocompatible match to the recipient or is mismatched, cordblood cells engraft and repopulate conditioned hosts as a treatment fora variety of congenital or acquired hematologic disorders. Even if thecord blood graft is mismatched with the recipient by two or more loci,the incidence and severity of GVHD is significantly less than thatobserved for transplantation of a similarly mismatched adult marrow ormobilized peripheral blood graft.

Limitations to a more widespread use of cord blood for transplantinclude the fact that only a limited number of HSC and progenitor cellsare present in a graft. Because most patients do not have a matchedsibling donor, most cord blood grafts are transplanted into mismatchedrecipients. Multiple studies report that the dose of cord blood cells ina graft is critical for patient survival when the graft comes from anunrelated donor. Transplant related mortality is reported as 20% inrecipients that obtained a cord blood graft with >1.7×10⁵ CD34+ cells/kgversus 75% in those receiving fewer CD34+ cells in the graft. Finding amethod to effectively expand cord blood HSC ex vivo to increase thenumber of cells in a graft, would be a major advance for clinicaltransplantation and would have a significant commercial market.

Approaches to cord blood HSC expansion have not been impressive. In moststudies, addition of a variety of growth factors to cord bloodmononuclear cells or isolated CD34⁺ cells has been correlated withincreases in total cell numbers, colony forming unit cell (CFC) numbers,and in short-term progenitor cell engraftment in immunodeficient(NOD/SCID) mice or fetal sheep. However, few approaches have beeneffective in increasing the number of HSC as measured by SCIDrepopulating cells (SRC) frequency in NOD/SCID mice or long-termengraftment in fetal sheep. The results of using expanded cord blood HSCin human patients have been disappointing.

SUMMARY OF THE INVENTION

A single-cell colony assay was developed to describe a novel hierarchyamong mammalian endothelial progenitor cells (EPCs) isolated fromperipheral blood and umbilical cord and from endothelial cells isolatedfrom umbilical or adult blood vessels. A distinct population ofprogenitor cells from human, bovine, porcine and rat biological sampleswas identified based on clonogenic and proliferative potential.

Endothelial progenitor cells (EPCs) were isolated from adult peripheraland umbilical cord blood and expanded exponentially ex vivo. Incontrast, human umbilical vein endothelial cells (HUVECs) or humanaortic endothelial cells (HAECs) derived from vessel walls are widelyconsidered to be differentiated, mature endothelial cells (ECs) and areutilized as “controls” for EPC studies. However, similar to adult andcord blood derived EPCs, HUVECs and HAECs derived from vessel walls canbe passaged for at least 40 population doublings in vitro. Utilizing anovel single cell deposition assay, which discriminates EPCs based ontheir proliferative and clonogenic potential, EPCs were found to residein HUVECs or HAECs. A single cell clonogenic assay was developed todefine a novel hierarchy of EPCs based on their proliferative andclonogenic potential. A complete hierarchy of EPCs was identified inHUVECs and HAECs derived from vessel walls and discriminated by theirclonogenic and proliferative potential. Diversity of EPCs exists inhuman vessels and provides a conceptual framework for determining boththe origin and function of EPCs in maintaining vessel integrity. EPCsare therefore readily obtained for clinical use e.g. grafts, either fromperipheral blood or from biopsies of human vessels.

Isolated endothelial colony forming cells have the followingcharacteristics:

(a) express cell surface antigens that are characteristic of endothelialcells, such as CD31, CD105, CD146, and CD144;

(b) do not express cells surface antigens that are characteristic ofhematopoietic cells, such as CD45 and CD14;

(c) ingest acetylated LDL; and

(d) form capillary-like tubes in MATRIGEL® (extracellular matrixproteins).

The isolated cells classified as HPP-ECFC also

(a) replate into at least secondary colonies of at least 2000 cells whenplated from a single cell;

(b) exhibit high proliferation;

(c) proliferate from a single cell; and

(d) express high levels of telomerase, at least 34% of that expressed byHeLa cells. HPP-ECFC also display a high nuclear to cytoplasmic ratiothat is >0.8, cell diameters <22 microns, and at least 10⁷ progenyderive from a single cell.

A method of isolating endothelial colony cells includes steps of:

(a) culturing cells from a biological sample on supports coated withextracellular matrix proteins;

(b) selecting cells that adhere to the supports and form replatablecolonies; and

(c) selecting single cells from the colonies.

The biological sample may be mammalian cord blood, or blood vessel.Human, bovine, porcine and rat sources are suitable. A single cell assayfor types of endothelial cells includes the steps of:

(a) cell sorting of biological samples using a specific sorting method;

(b) culturing the single sorted cells on extracellular matrix proteinunder defined conditions; and

(c) enumerating specific colony sizes, morphology, and proliferativepotential to determine the type of endothelial cell e.g. HPP-ECFC.

A method of enriching for HPP-ECFC includes the steps of:

(a) cell sorting of biological samples using a specific sorting method;and

(b) culturing of single sorted cells on extracellular matrix proteinsunder defined conditions.

A method for expanding hematopoietic stem cells ex vivo, include thesteps of:

(a) culturing HPP-ECFC cells on collagen coated solid supports; and

(b) expanding hematopoietic stem cells (HSC) by co-culturing withHPP-ECFC cells wherein the HPP-ECFCs are derived from human cord bloodcells and the HSC cells are derived from human bone marrow.

A method for improving the percentage of hematopoietic stem cells in agraft in a mammal, includes the step of:

(a) co-culturing human bone marrow cells with cord blood HPP-ECFC toform a product; and

(b) transplanting a suitable amount of the product into the mammalwherein the cells are CD45+ cells derived from human bone marrow, andthe mammal is a NOD-SCID mouse.

Cord blood high proliferative potential-endothelial colony forming cells(HPP-ECFCs) in co-culture with autologous or unrelated cord blood,mobilized adult peripheral blood, or marrow-derived HSC expands thenumber of HSC cells and results in an increase in HSC and an increase inHSC repopulating activity leading to higher levels of engraftment in arecipient subject.

Use of a feeder layer of cells derived from high proliferativepotential-endothelial colony forming cells (HPP-ECPCs) from humanumbilical cord blood, stimulates growth and survival of repopulatinghematopoietic stem and progenitor cells. Stimulation of growth andsurvival was determined by increased numbers of progenitor cells in invitro culture and increased levels of human cell engraftment in theNOD/SCID immunodeficient mouse transplant system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. Isolation of endothelial progenitor cell colonies derivedfrom adult peripheral and umbilical cord blood. (FIG. 1A) Number of EPCcolonies isolated per 20 ml of adult peripheral (AB) and umbilical cord(CB) blood. Results represent the average number of EPC colonies ±SEM of18 independent experiments for adult donors and 13 independentexperiments for cord blood samples. *P<0.0001 by Student paired t test.(FIG. 1B) Time of initial EPC colony appearance after culture initiationfrom equivalent volumes of adult peripheral (AB) and umbilical cord (CB)blood. Results represent the average number of days before initial EPCcolony appearance ±SEM of 18 independent experiments for adult donorsand 13 independent experiments for cord blood donors. *P<0.0001 byStudent paired t test. (FIG. 1C) Representative low and high powerphotomicrographs of endothelial cell colonies derived from adult blood.(FIG. 1D) Low and high power photomicrographs of endothelial cellcolonies derived from CB. (FIG. 1E) Endothelial cell monolayers derivedfrom adult endothelial cell colonies at low and high powermagnification. (FIG. 1F) Endothelial cell monolayers derived from CBendothelial cell colonies at low and high magnification. Scale bar inphotomicrographs represents 100 μm (left column FIGS. 1C-1F) and 10 μm(right column FIGS. 1C-1F).

FIGS. 2A-2E. Phenotypic and functional analysis of adult and cord bloodEPC-derived endothelial cells. (FIGS. 2A-2B) Immunophenotyping of cellmonolayers derived from either adult (FIG. 2A) or cord (FIG. 2B) EPCcolonies by fluorescence cytometry. Both cord blood and adultEPC-derived cells express CD31, CD141, CD105, CD 146, CD 144, vWF, andFlk-1, but do not express CD45 and CD 14. Some cord blood and adultcells express CD34, CD133, and CD117. Shown is representative data from18 independent experiments utilizing different adult cell monolayers and13 independent experiments using different cord blood cell monolayerswith similar results. Isotype controls are overlayed in gray on eachhistogram for each surface antigen tested. (FIG. 2C) Adult and cordblood EPC-derived endothelial cells incorporate DiI-Ac-LDL (50×magnification). A representative photomicrograph is shown for adult andcord blood EPC-derived endothelial cells, which have taken up DiI-Ac-LDL(red) and also stained with DAPI (blue). Shown is representative datafrom 18 independent experiments utilizing different adult cellmonolayers and 13 independent experiments using different cord bloodcell monolayers with similar results. Scale bar in photomicrographsrepresents 100 μm. (FIG. 2D) Adult and cord blood EPC-derivedendothelial cells genetically engineered to express enhanced greenfluorescence protein (EGFP) plated in MATRIGEL® (extracellular matrixproteins) for formation of capillary-like structures (50×magnification). Shown is representative data from 18 independentexperiments utilizing different adult cell monolayers and 13 independentexperiments using different cord blood cell monolayers with similarresults. Scale bar in photomicrographs represents 100 μm. (FIG. 2E)Adult and cord blood EPC-derived endothelial cells upregulate the cellsurface expression of vascular cell adhesion molecules (VCAM-1) inresponse to either rhTNF-α or rhIL-1. Shown is representative data from18 independent experiments utilizing different adult cell monolayers and13 independent experiments using different cord blood cell monolayerswith similar results. The isotype control for VCAM-1 is overlayed ingray on each histogram.

FIG. 3. Representative photomicrographs of morphologically distinctlydifferent secondary colonies, which formed seven days after adult andcord blood (CB) EPC-derived endothelial cells were re-plated at low celldensity.

FIGS. 4A-4D. Growth kinetics of the endothelial cell progeny derivedfrom cord and adult endothelial progenitor cell colonies. (FIG. 4A) Exvivo expansion of adult (AB) and cord (CB) blood EPC-derived endothelialcells harvested from mononuclear cells. Black boxes represent total cellnumber at each passage. Cells uniformly expressed the endothelial cellsurface antigens shown above (FIGS. 2A-2B) and not the hematopoieticcell specific antigens, CD45 and CD14 at each passage. A representativegrowth curve for cord blood and adult EPC-derived endothelial cells isshown. Eleven other cord blood and adult endothelial cell monolayersderived from different donors showed similar growth kinetics. (FIGS.4B-4C) Population doubling times (PDT) and cumulative populationdoubling levels (CPDL) of cord (CB) blood and adult (AB) EPC-derivedendothelial cells during 60 days of culture. Results represent theaverage number of PDTs and CPDLs ±SEM of 6 independent experiments.*P<0.01 by Student paired t test. (FIG. 4D) DNA synthesis of cord (CB)blood and adult (AB) EPC-derived endothelial cells. Early passage (1-2)cord blood EPC-derived endothelial cells demonstrate increased DNAsynthesis in response to 10% FBS, rhVEGF, and rhbFGF compared to adultcells. Results represent the average of 4 independent experiments usingendothelial cells derived from different donors. *P<0.01 by Student'spaired t test.

FIGS. 5A-5E. Quantitation of the clonogenic and proliferative potentialof single cord blood and adult endothelial cells derived from EPCcolonies. (FIG. 5A) Schematic of single cell assays using endothelialcells derived from either adult or cord EPC colonies. (FIG. 5B) Thepercentage of single adult (AB) or cord (CB) blood EPC-derivedendothelial cells undergoing at least one cell division after 14 days ofculture. Results represent the average of 5 independent experimentsusing single endothelial cells derived from different donors. *P<0.01 byStudent's paired t test. (FIG. 5C) Average number of cell progenyderived from a single adult (AB) or cord (CB) blood EPC-derivedendothelial cell after 14 days in culture. Results represent the averageof 5 independent experiments using single endothelial cells derived fromdifferent donors. *P<0.01 by Student's paired t test. (FIG. 5D) Percentof dividing single cells giving rise to a colony with the number ofcells in the quantitative ranges shown (HPP, LPP, clusters) (FIG. 5E)Representative photomicrographs (50× magnification) of the differentendothelial cell clusters (<50 cells), LPP (about 51-2000 cells), andHPP (about 2000- to >10,000 cells) derived from a single cord blood oradult EPC-derived endothelial cell. Results are representative of 4other independent experiments utilizing cells from different donors.Scale bar in photomicrographs represents 100 μm. LPP-ECFC (51-2000cells) and HPP-ECFC (2000>10,000 cells) ranges are approximations.

FIGS. 6A-6E. Replating potential and long-term culture of the cellprogeny derived from a single cord blood or adult EPC-derivedendothelial cell. (FIG. 6A) Percent of the cell progeny derived from asingle cord (CB) blood or adult (AB) EPC-derived endothelial cell, whichformed secondary colonies or rapidly grew to cell confluence after 7days of culture in a 24 well tissue culture plate. Results represent theaverage ±SEM of 4 independent experiments using cells derived from 4different donors. *P<0.01 by Student's paired t test. (FIG. 6) Arepresentative photomicrograph (50× magnification) of the secondaryendothelial cell colonies or confluent cell monolayers derived from thecell progeny of a single plated cord blood derived endothelial cell in a24 well plate after 7 days in culture. Scale bar in photomicrographsrepresents 100 μm. (FIG. 6C) Growth kinetics of the cell progeny of 11single plated endothelial cells isolated from three different cord blooddonors in long-term culture. Black boxes represent the total number ofcells at each passage. (FIG. 6D) Telomerase activity of 1000 earlypassage adult (lanes 1-4) and cord (lanes 5-8) blood EPC-derivedendothelial cells isolated from different donors. Adult and cord cellswere tested at a CPDL of 15. P indicates telomerase activity in 1000HeLa cells, which were used as a positive control, and N indicates anegative control. The average level of telomerase activity in the adultsamples was 4±4% and of the cord blood samples 34±10% of the telomeraseactivity of the HeLa cells (FIG. 6E) Comparison of telomerase activityof early and late passage adult (FIG. 6A) and cord (FIG. 6C) bloodEPC-derived endothelial cells. PD indicates the cumulative populationdoubling level of the cells tested. P indicates telomerase activity inHeLa cells, which were used as a positive control. N indicates anegative control. Three other experiments utilizing early and latepassage cord blood and adult EPC-derived endothelial cells from threedifferent donors showed similar results.

FIG. 7. Model of an endothelial progenitor cell hierarchy based on theproliferative and clonogenic potential of discrete populations ofprogenitor cells. High proliferative potential-endothelial colonyforming cells (HPP-ECFC) are large colonies arising from single cellsthat form at least secondary colonies upon replating. HPP-ECFC give riseto all subsequent stages of endothelial progenitors in addition toreplating into secondary HPP-ECFCs. Low proliferativepotential-endothelial colony forming cells (LPP-ECFC) arising fromsingle cells form colonies which contain greater than 50 cells, but donot form secondary colonies of LPP-ECFCs upon replating. Endothelialcell clusters (EC-clusters) can arise from a single cell but containless than 50 cells, which are typically larger compared to the smallercells (See FIGS. 10A-10D) found in HPP-ECFC and LPP-ECFC colonies.Mature terminally differentiated endothelial cells do not divide.

FIGS. 8A-8C. Immunophenotypic analysis of endothelial cells from cordblood-EPC, HUVECs, and HAECs. (FIGS. 8A-8C) Immunophenotyping of cellmonolayers derived from cord blood EPCs (FIG. 8A) umbilical veins (FIG.8B) or human aortas (FIG. 8C) by fluorescence cytometry. Cord bloodEPC-derived ECs, HUVECs and HAECs express CD31, CD141, CD105, CD146,CD144, vWF, and Flk-1, but do not express CD45 and CD 14. Shown isrepresentative data from 5 independent experiments utilizing 5 differentcord blood EC monolayers, 5 different HUVEC samples and 5 different HAECsamples with similar results. Isotype controls are overlayed in white oneach histogram for each surface antigen tested.

FIGS. 9A-9F. Quantitation of the clonogenic and proliferative potentialof single endothelial cells derived from cord blood-EPC colonies,HUVECs, and HAECs. (FIG. 9A) The percentage of single cord blood (CB)EPC-derived ECs, HUVECs, or HAECs undergoing at least one cell divisionafter 14 days of culture. Results represent the average of fiveindependent experiments using single ECs derived from different donors.(FIG. 9B) percent of dividing single cells in an individual well—SeeFIG. 5B. Percent of single CB EPC-derived EC, HUVEC, or HAEC giving riseto colonies of cells (as classified) in an individual well after 14 daysof culture. *P<0.01 by Student's paired test for comparison of a singleCB-derived EC versus either a single HUVEC or HAEC. (FIG. 9C)Representative photomicrographs (50× magnification) of the different ECclusters (<50 cells) or colonies (>50 cells) derived from a single cordblood EPC-derived EC, HUVEC, or HAEC. Results are representative of fourother independent experiments utilizing cells from different donors.Scale bar in photomicrographs represents 100 μm. (FIG. 9D) Percent ofthe cell progeny derived from a single cord blood EPC-derived EC, HUVEC,or HAEC, which formed secondary colonies or rapidly grew to cellconfluence after seven days of culture in a 24 well tissue cultureplate. Results represent the average ±SEM of 4 independent experimentsusing cells derived from four different donors. *P<0.01 by Student'spaired t test for comparison of a single CB-derived EC versus either asingle HUVEC or HAEC. (FIG. 9E) A representative photomicrograph (50×magnification) of the secondary EC colonies or confluent cell monolayersderived from the cell progeny of a single plated cord blood EPC-derivedEC, HUVEC, or HAEC in a 24 well plate after seven days in culture. Scalebar in photomicrographs represents 100 μm. (FIG. 9F) Telomerase activityin an HPP-ECFC (HPP) and LPP-ECFC (LPP) colony derived from HUVECs. Pos.indicates telomerase activity in HeLa cells, which were used as apositive control, and Neg. indicates a negative control. Results arerepresentative of four other independent experiments. Similardifferences in telomerase activity were observed between HPP-ECFC andLPP-ECFC colonies isolated from cord blood ECs and HAECs.

FIGS. 10A-10D. Monochromatic images of (FIG. 10A) HPP-ECFC, (FIG. 10B)LPP-ECFC, (FIG. 10C) endothelial clusters, and (FIG. 10D) maturedifferentiated endothelial cells. The HPP-ECFC are small cells (nucleardiameter 8-10 microns) with minimal cytoplasmic spreading (diametersvary from 12-22 microns) with nuclear to cytoplasmic ratio >0.8.LPP-ECFC are more heterogenous in size but are larger than HPP-ECFC.LPP-ECFC nuclei vary in size from 10.5-12.5 microns and have morecytoplasmic spreading (varying from 25-60 microns) with a ratio >0.4 but<0.5. Endothelial clusters are nearly mature endothelial cells withnuclei that vary from 13.0-16.5 microns and have cytoplasmic diametersthat vary from 65-80 microns and nuclear to cytoplasmic ratios of >0.2but <0.3. Mature differentiated endothelial cells are large very wellspread cells with nuclear diameters that range from 17.0-22.0 micronsand cytoplasmic diameters from 85-105 microns and nuclear to cytoplasmicratios similar to endothelial clusters. Therefore, HPP-ECFC are verydistinctly smaller than any of the other EPC and quite smaller than themature endothelial cells.

FIG. 11. The percent of dividing single plated bovine, porcine, and rataortic endothelial cells giving rise to colonies of distinct classifiedsizes.

FIGS. 12A-12C. Percent chimerism of human cells (CD45 positive) detectedby flow cytometry in the peripheral blood of NOD-SCID mice eight weeksafter (FIG. 12A) transplantation of human bone marrow derived CD34+cells injected on the same day of isolation; (FIG. 12B) cultured for 7days with maximal stimulating concentrations of G-CSF, TPO, SCF, orFlt-3; (FIG. 12C) or co-cultured with cord blood HPP-ECFC for 7 dayswhere the gated cells stain positive for the human CD45 antigen; graphsare representative of 3 independent experiments with similar results.

DETAILED DESCRIPTION OF THE INVENTION

A new endothelial cell progenitor named a high proliferativepotential-endothelial colony forming cell (HPP-ECFC) displays highproliferative potential (up to 100 population doublings compared to20-30 doublings in adult blood EPC. HPP-ECFC were not only isolated fromcord blood but from umbilical and adult blood vessels. HPP-ECFC cellscan be replated at a single cell level and the majority of cellsproliferate with regeneration of at least secondary HPP-ECFCs. (FIG. 7)Unexpectedly, HPP-ECFC colony formations are present in cord blood butnot in adult peripheral blood (FIG. 5D). Further, monolayers of cordblood endothelial cells derived from HPP-ECFCs demonstrate a 2.5-folddecrease in population doubling times (PDT) and at least a 2-foldincrease in cumulative population doubling levels (CPDL) compared toadult LPP-ECFCs (during the same time of culture ex vivo). In contrastto other populations of endothelial progenitor cells isolated from cordblood utilizing different methodologies, cord blood HPP-ECFC progenyuniformly express endothelial cell antigens and not hematopoieticspecific cell antigens. Thus, HPP-ECFC are enriched in human umbilicalcord blood and were not found in adult peripheral blood. HPP-ECFC werealso found in endothelial cells derived from mammalian blood vesselse.g. umbilical vein and human aortic vessels. (FIGS. 1A-1F) TheseHPP-ECFCs appear in cultures of freshly plated cord blood mononuclearcells within 10 days, whereas adult blood LPP-ECFCs rarely appear before14 days after plating.

Further, cord blood colonies consistently appeared larger compared toadult colonies (FIGS. 1A-1F). There were distinct differences in thesize, frequency and time of appearance between adult and cord bloodendothelial cell colonies. (FIGS. 1A-1F, 3, and 10A-10D) Theseobservations show that cord blood EPCs are composed of HPP-ECFC,LLP-ECFC, and clusters, whereas adult blood EPCs are composed ofLPP-ECFCs and clusters.

The complete hierarchy of HPP-ECFC, LPP-ECFC, clusters and matureendothelial cells can be isolated from any blood vessel in a livingmammalian donor.

EPCs can be identified using similar terminology to that utilized fordefining hematopoietic cell progenitors (FIG. 7). HPP-ECFCs give rise toall subsequent stages of endothelial progenitors in addition toreplating into secondary HPP-ECFCs. Low proliferativepotential-endothelial colony forming cells (LPP-ECFC) arising fromsingle cells form colonies, which contain greater than 50 cells, but donot form at least secondary LPP-ECFC colonies upon replating. They dogive rise to endothelial cell clusters (less than 50 cells). Finally,endothelial cell clusters can arise from a single cell but contain lessthan 50 cells, and do not replate into colonies or clusters.

Hematopoietic stem and progenitor cells are enriched in umbilical cordcompared to adult peripheral blood. One intriguing observation was thatEPCs were also enriched in umbilical cord blood compared to adultperipheral blood. Further, cord blood derived EPCs contain high levelsof telomerase activity, which may account for the observation that thesecells can be expanded for at least 100 population doublings withoutobvious signs of cell senescence. At the single cell level, some cordblood EPC-derived cells can be expanded 10⁷ to 10¹² fold. To ourknowledge, no other primary mammalian endothelial progenitor or maturecell has been identified with similar growth characteristics orclonogenic capacity.

Characterization of Endothelial Cell Colonies (EPCs) Isolated from HumanUmbilical Cord Blood

Cells were expanded in culture while maintaining an endothelial cellphenotype. In contrast to previously described endothelial progenitorcells isolated from cord blood, the present invention relates that cellsisolated from human umbilical cord blood, i.e. cord blood HPP-ECFCs andprogeny, can be cultured for at least 100 population doublings andexpanded exponentially even when beginning with a single cell. Further,these cells do not express the hematopoietic cell specific surfaceantigens, CD45 and most also do not express CD14, and do not formhematopoietic cell colonies in methycellulose assays. In addition, theHPP-ECFC progeny rapidly form vessels in MATRIGEL® (extracellular matrixproteins), upregulate VCAM-1 in response to either IL-1 or TNFαstimulation, and express endothelial cell specific antigens, whichconfirms their endothelial cell identity. These cells were designatedhigh proliferative potential-endothelial colony forming cells(HPP-ECFCs).

Cord blood HPP-ECFCs demonstrate greater replicative kinetics comparedto adult blood (which are composed of LPP-ECFC, clusters, and matureendothelial cells). While endothelial progenitors are reported toexpress AC133, CD34, and Flk1, HPP-ECFC progeny and EOC progeny displaysimilar frequencies of cells expressing AC133, CD34, and Flk1 antigensand, therefore, these cell surface markers do not permit discriminationof cells with differing proliferative potentials.

Endothelial outgrowth cells appear two to four weeks after culture ofMNCs isolated from adult peripheral blood and are characterized by theirexponential growth in vitro (however, EOC display lower levels oftelomerase, do not replete into secondary LPP-ECFC, and reachreplicative senescence long before TPP-ECFC). In contrast, HPP-ECFCgenerated from human umbilical cord blood MNCs, emerged five to ten daysafter culture of cord blood MNCs in complete EGM-2 media on tissueculture plates coated with type I collagen. Discrete adherent cellcolonies appeared and displayed the “cobblestone” morphology ofendothelial cells (FIGS. 1C-1F). The morphology and appearance of thecolonies was similar to, but distinct from, that previously describedfor adult peripheral blood derived EOC colonies and clearly distinctfrom adherent circulating endothelial cells or macrophages. The HPP-ECFCcolonies are large and are composed of a mixture of small round, longthin, and large flattened round cells whereas the EOC are nearlyhomogenously composed of long thin cells. The colony-derived cells weresubcultured and expanded cells derived from these colonies were used forimmunophenotyping, functional testing and measurement of growthkinetics. After initial passage, the cells formed monolayers of spindleshaped cells with “cobblestone” morphology. Immunophenotyping revealedthat the cells uniformly expressed the endothelial cell surfaceantigens, CD31, CD141, CD105, CD146, CD144, vWF, and flk-1 (FIGS.2A-2B). The cells did not express the hematopoietic cell surfacespecific antigens, CD45 and CD 14, confirming that the monolayers werenot contaminated with hematopoietic cells. (FIGS. 1A-1F, 3, and10A-10D).

Confirming that the monolayers derived from the adherent colonies wereendothelial cells, the cells ingested acetylated-low density lipoprotein(Ac-LDL) or (Dil-AC-LDL). These cellular functions are characteristic ofendothelial cells. Cells subcultured from the adherent coloniesuniformly incorporated Ac-LDL, formed vessels in MATRIGEL®(extracellular matrix proteins) after seeding varying numbers of cells,and upregulated VCAM-1 in response to both rhTNF-α or rhIL-1 stimulation(FIGS. 2C-2E).

The growth kinetics of cord blood HPP-ECFC and progeny were measured asa function of time. Strikingly, cord blood HPP-ECFC and progeny could beexponentially expanded in culture for at least 100 population doublingswithout signs of senescence, and the number of cells increased 10²⁰ foldover a period of 100 days in culture. A representative growth curve ofcord blood endothelial cells, illustrates the proliferative potential ofthese cells in vitro. Thus, based on immunophenotyping, functionaltesting, and an analysis of growth kinetics, it was shown that coloniesof endothelial cells (designated HPP-ECFCs) can be uniquely culturedfrom cord blood MNCs and passaged into confluent monolayers ofexponentially expandable endothelial cells.

HPP-ECFC Colonies are Present in Human Umbilical Cord Blood but not inAdult Peripheral Blood

50-100 milliliters of peripheral blood was collected from healthy adultdonors or from umbilical cords of normal term infants and isolated MNCs.Cells were seeded into tissue culture plates coated with extracellularmatrix molecules in complete EGM-2 media and observed for colonyformation over the next one to six weeks. The number of colonies perequivalent volume of blood was increased 15 fold in cord blood comparedto adult peripheral blood (Table I, FIGS. 1A and 1B). Similardifferences in colony formation were also observed when equivalentnumbers of cord and adult MNCs were plated. Although adult EOC coloniestypically formed between 2-4 weeks after initiation of culture, cordblood HPP-ECFC colonies appeared within 5-10 days. Finally,immunophenotyping of the cells isolated from endothelial colonies fromboth cord blood and adult peripheral blood by flow cytometry revealedthat the colony-cells uniformly expressed the endothelial cell surfaceantigens, CD31, CD105, CD146, CD144, vWF, and flk-1 and not thehematopoietic cell surface antigens CD45 and CD 14, confirming theirendothelial cell identity. Immunophenotyping of the adult EOCs wasconsistent with previously published studies. Thus, endothelial colonyforming cell HPP-ECFC are present in cord blood and represent adifferent cell type compared to adult peripheral blood EOCs (whichrepresent LPP-ECFCs).

The Proliferative Rate of Cord Blood HPP-ECFCs is Greater than AdultBlood EOCs

Given the differences in the frequency and the time of appearance ofcolony formation of cells from cord blood compared to adult peripheralblood, a question was whether there were differences in theproliferative kinetics of adult and cord blood cells. Early passagemonolayers of HPP-ECFC were established from cord blood and EOCestablished from adult peripheral blood, and cells were cultured incomplete EGM-2 media on type I collagen coated plates. Input cellnumbers were counted for determination of population doubling times(PDT) and cumulative population doubling levels (CPDL) in long-termcultures, which were measurements used to quantitate and to compare theproliferative kinetics of cord blood and adult blood derived cells.Cells were cultured for at least 10 passages to accurately quantitatethe PDT and CPDL. Results testing multiple cell lines from differentdonors, showed that there was a 2.5 fold decrease in the PDT of cordblood HPP-ECFCs compared to adult EOC controls. Further, consistent witha decrease in PDT, culture of cord blood cells demonstrated asignificant increase in CPDLs compared to serial passage of adult EOCs.Thus, although both cord and adult cells can be expanded in culture, theproliferative potential of cord blood HPP-ECFC and progeny is greatercompared to adult EOC. Cord blood derived HPP-ECFC also demonstrategreater proliferative potential at the single cell level compared toadult blood EOCs.

The proliferative and clonogenic capacity of individual cord bloodHPP-ECFC-derived endothelial cells or adult EOCs at the single celllevel was determined. A novel experimental method was designed toquantitate the proliferative and clonogenic capacity of single cordblood HPP-ECFC-derived endothelial cells and adult EOCs.

Early passage cord blood HPP-ECFC-derived endothelial cells or adult EOCprogeny were initially transduced with a retrovirus encoding a greenfluorescent protein (GFP) and selected for expression of GFP.Transduction efficiency of both cord and adult endothelial cells wasgreater than 95%. Following selection, one GFP expressingHPP-ECFC-derived endothelial cell or adult EOC-derived endothelial cellwas plated by fluorescent cytometry sorting (using a sorting nozzle witha diameter ≥100 microns and a sheath flow pressure of ≤9 pounds persquare inch) into one well of a 96 well tissue culture plate coated withtype I collagen and filled with 200 μl of EGM-2 media. Immediatelyfollowing placement, individual wells were examined to ensure that onlyone endothelial cell had been placed into each well. Endothelial cellswere then cultured for 14 days, and one half of the media was changedevery 4 days with fresh EGM-2 media. At the end of 14 days, the numberof GFP expressing endothelial cells was counted.

The number of single cells undergoing at least one cell division wassignificantly greater for cord blood HPP-ECFC-derived endothelial cellscompared to adult EOC-derived endothelial cells. In scoring the numberof cells in each well at the end of 14 days, it was clear that singlecord blood HPP-ECFC-derived endothelial cells divided more and producedlarger colonies compared to adult EOC-derived endothelial cells. Becauseof differences in the capacity of single cord blood HPP-ECFC-derivedendothelial cells to divide and form colonies compared to adultEOC-derived endothelial cells, the number of cells in each well, whichdemonstrated at least one cell division, were counted. Although, most ofthe single adult EOC-derived endothelial cells (which had divided),produced clusters of between 2 and 50 cells, some did give rise tosecondary colonies of up to 500 cells, but only a single colony of >2000cells arose from any of the single sorted adult EOC-derived endothelialcells. However, greater than 60% of the cord blood HPP-ECFC-derivedendothelial cells (which had divided), formed well circumscribedsecondary colonies consisting of at least 2000 cells, and numeroussingle sorted cells gave rise to colonies composed of >10,000 cells (tothe inventors' knowledge, no adult EOC-derived endothelial cells everproduced such a colony).

Secondary cell colonies derived from either single adult EOC-derived orcord blood HPP-ECFC-derived endothelial cells were serially replated todetermine if these cells could form more colonies. Secondary coloniesderived from single adult EOC-derived endothelial cells never gave riseto tertiary colonies after replating in 24 well or 6 well type Icollagen coated tissue culture plates in multiple independentexperiments. Single cells plated remained quiescent and did notproliferate. However, most of the secondary colonies derived from singlecord blood HPP-ECFC-derived endothelial cells, which produced greaterthan 2000 cells, could be replated under the same experimentalconditions to form tertiary endothelial colonies. Single primary cordblood HPP-ECFC-derived endothelial cells can produce secondary colonies,which can be subsequently serially passaged to produce from 10⁷-10¹²endothelial cells.

Given the similarities of this unique and newly identified population ofcord blood derived endothelial colony forming cells to the hematopoietichigh proliferative potential-colony forming cells (HPP-CFC; the mostprimitive multipotent hematopoietic progenitor that can be cultured inan in vitro clonogenic assay) these cells are named “high proliferativepotential-endothelial colony forming cells (HPP-ECFC)”. In summary,these cells are different from the previously described adult EOCs inthe following ways: (1) HPP-ECFCs have higher proliferative kineticswhen cultured under the same experimental conditions as adult EOCs, (2)HPP-ECFCs appear at earlier timepoints in culture from plated cord bloodMNCs compared to adult EOCs derived from plated adult peripheral MNCs,(3), HPP-ECFCs have higher clonogenic potential at the single cell levelcompared to adult EOCs. (4) HPP-ECFCs can be serially replated to format least secondary HPP-ECFC colonies while/whereas adult EOCs do notdisplay this potential, and HPP-ECFC display high levels of telomerase.

Growth Kinetics of EPC-Derived Cord Blood and Adult Endothelial Cells

Progenitor cells of different lineages are defined and discriminated bytheir clonogenic and proliferative potential. Because of the differencesin cord blood and adult EPC colony formation, the proliferative kineticsof EPC-derived cord blood and adult endothelial cells were compared.Initially cells derived from cord blood and adult endothelial cellcolonies were plated at limiting cell dilutions to test whether thecells would form secondary colonies and grow to confluence.Interestingly, the cell progeny derived from both adult and cord bloodEPC colonies formed secondary cell colonies of various sizes beforegrowing to confluence (FIG. 3). However, colonies derived from cordblood EPC-derived cell progeny were consistently larger and containedsmaller cells compared to adult colonies (FIG. 3).

Cell monolayers were serially passaged to determine the proliferativepotential of EPC-derived cord blood and adult endothelial cells.Remarkably, cord blood EPC-derived cells could be expanded for at least100 population doublings without obvious signs of senescence. Incontrast, adult EPC-derived cells could be passaged for only 20-30population doublings (FIG. 4A). To quantitate and compare theproliferative kinetics of cord blood and adult EPC-derived cells, thepopulation doubling times (PDT) and cumulative population doublinglevels (CPDL) were calculated during a defined time in culture (60days). There was a 2.5 fold decrease in the PDT and a 1.5 fold increasein the CPDLs of cord blood EPC-derived cells compared to adultEPC-derived cells (FIGS. 4B and 4C). The PDT and CPDL of adult EPCs wassimilar to two recent reports, which tested the proliferative kineticsof EPC-derived cells isolated from healthy adult donors.

The proliferation of cord blood and adult EPC-derived cells in responseto either rhVEGF or rhbFGF stimulation, which are two endothelial cellmitogens were compared. Cord blood and adult EPC-derived cells wereserum starved and then cultured in the presence or absence of eitherrhVEGF or rhbFGF. Cells were cultured for 16 hours, and pulsed withtritiated thymidine before harvest to measure DNA synthesis. Cord bloodEPC-derived cells displayed greater DNA synthesis in response to eitherrhVEGF or rhbFGF stimulation compared to adult EPC-derived cells (FIG.4D). Collectively, these results demonstrate that the proliferative rateand expandability of cord blood EPC-derived cells is greater than adultEPC-derived cells in both short and long term assays. Further, cordblood and adult EPC-derived endothelial cells form distinct cellcolonies of various sizes and morphology when plated at limitingdilution.

Quantitation of the Clonogenic and Proliferative Potential of SingleCord Blood and Adult Endothelial Cells Derived from EPC Colonies

Cord blood and adult EPC colonies yield cells with differentproliferative and clonogenic potential. However, a rigorous test for theclonogenic potential of a progenitor cell is to determine whether asingle cell will divide and form a colony in the absence of other cells.Therefore, an assay was developed to quantitate the proliferative andclonogenic potential of single cord blood and adult endothelial cellsderived from EPC colonies.

Cord blood and adult endothelial cells derived from the initial EPCcolonies were transduced with a retrovirus encoding EGFP and selectedfor EGFP expression. Following selection, one EGFP expressingendothelial cell was plated by FACS into one well of a 96 well tissueculture plate coated with type I collagen and filled with complete EGM-2media. Endothelial cells were cultured, and the number ofEGFP-expressing endothelial cells was counted at the end of 14 days asdisclosed herein. This method is illustrated in FIG. 5A.

Remarkably, the percentage of single cells undergoing at least one celldivision was increased five fold for cord blood endothelial cellscompared to adult cells (FIG. 5B). Further, the average number of cellprogeny derived from a single cord blood endothelial cell was 100 foldgreater compared to the number of cells derived from an individual adultcell (FIG. 5C). Greater than 80% of the single adult endothelial cellswhich divided gave rise to small colonies or clusters of cells rangingin number from 2-50 cells (FIG. 5D). However, some single adultendothelial cells did form colonies containing greater than 500 cells(FIG. 5D). In contrast, at least 60% of the single plated cord bloodendothelial cells which divided formed well-circumscribed coloniescontaining between 2,000 and 10,000 cells in the 14 day culture period(FIG. 5D). Photomicrographs of the size and morphology of the variousendothelial cell colonies and clusters of cells derived from a singlecord blood or adult cell are shown in FIG. 5E. These single cell studiesdemonstrate that there are different types of cord and adult EPCs, whichcan be discriminated by their proliferative and clonogenic potential,and that EPCs display a hierarchy of proliferative potentials similar tothe hematopoietic progenitor cell hierarchy.

The Cell Progeny of Single Cord Blood Endothelial Cells can be SeriallyReplated and Expanded Exponentially in Long-Term Cultures.

In the hematopoietic cell system, the most proliferative progenitor celltype is termed the high proliferative potential-colony forming cell(HPP-ECFC). The HPP-ECFC is defined by its ability to form large cellcolonies, which yield individual cells that have the potential to format least secondary colonies upon serial replating. The clonal progenyderived from a single plated cord blood or adult EPC-derived cell weretrypsinized, replated and cultured into 24-well tissue culture platesfor 7 days. After plating the clonal progeny of over 1000 single adultEPC-derived cells into 24 well plates, only one secondary colony wasdetected in the wells after 14 days of culture (FIG. 6A). In contrast,approximately one half (205 of 421) of the clonal progeny of singleplated cord blood EPC-derived cells formed secondary colonies or rapidlygrew to confluence in 24 well plates (FIG. 6A). A representativephotomicrograph of the secondary endothelial cell colonies or confluentcell monolayers derived from the progeny of a single cord bloodendothelial cell is shown in FIG. 6B. Since secondary colonies were notdetected in those wells that had rapidly grown to confluence in 5 days,a limiting dilution analysis was performed on the confluent monolayer.At least nine percent of the single cells plated from this monolayerformed an endothelial cell colony, containing greater than 100 cells.This result verifies that individual cells derived from cord blood EPCsare capable of forming secondary colonies.

The long-term proliferative potential of the cells derived from a singleplated cord blood EPC-derived endothelial cell was tested. Secondarycolonies or confluent cell monolayers derived from single cord bloodendothelial cells were serially passaged into progressively largertissue culture plates. The cell progeny of 11 single endothelial cells,originally derived from three different cord blood donors were tested.Single cord blood endothelial cells yielded at least 10⁷ cells inlong-term culture. (FIG. 6C). The average CPDL of the eleven single cordblood endothelial cells tested was 30.8. Thus, a population of highproliferative EPCs in cord blood, which form secondary and tertiarycolonies.

EPC-Derived Cord Blood Endothelial Cells Contain High Levels ofTelomerase Activity.

Endothelial cells derived from cord blood EPCs were serially passagedbeyond Hayflick's limit for at least 100 population doublings (FIG. 4A).The only other reported primary endothelial cells with similar growthkinetics are those genetically engineered to overexpress telomerase.Thus, telomerase activity was measured in cord blood and adultEPC-derived cells as a potential molecular explanation for thedifferences in their growth kinetics. Both early and late passage cordblood EPC-derived progeny display significantly elevated levels oftelomerase activity compared to adult EPC-derived cells, reminiscent ofthe previously described primary endothelial cells lines, whichoverexpress telomerase (FIGS. 6D-6E). Thus, consistent with extensiveproliferative potential, cord blood EPC-derived cells retain high levelsof telomerase activity (34±10% of the telomerase activity of an equalnumber of HeLa cells) with serial passage in culture.

Expansion of Repopulating Stem and Progenitor Cells Ex Vivo

Cord blood high proliferative potential-endothelial colony forming cell(HPP-ECFC) in co-culture with autologous or unrelated cord blood,mobilized adult peripheral blood, or marrow-derived HSC, expands thenumber of HSC cells and results in an increase in HSC and an increaseHSC repopulating activity leading to higher levels of engraftment in arecipient subject.

Co-culture of HPP-ECFC from cord blood with human HSC increaseshematopoietic progenitor cell numbers and enhances engraftment of humanhematopoietic cells in NOD/SCID mice, an assay for in vivo measure ofhuman HSC function.

Human cord blood HPP-ECFC-derived endothelial cells co-cultured withhuman cord blood or mobilized adult peripheral blood CD34⁺CD38⁻ cells(enriched in HSC activity) for up to 7 days (with added cytokines)results in an enhancement in human CD45⁺ cell engraftment in sublethallyirradiated NOD/SCID mice by >100 fold (FIGS. 12A-12C).

A method of collection, isolation, and expansion of the HPP-ECFC and theparticular method for co-culturing the HPP-ECFC with human stem cellsare novel. HPP-ECFC can be collected from any cord blood sample,expanded, frozen, and stored. These cells can then be thawed, expanded,and used in co-culture to expand human cord blood, marrow-derived, ormobilized adult peripheral blood stem and progenitor cell samples. Theexpanded product can then be used for transplantation purposes (afterregulatory agency approval).

Materials and Methods

Adult Peripheral and Umbilical Cord Blood Samples

Fresh blood samples (50-100 ml) were collected by venipuncture andanticoagulated in citrate phosphate dextrose solution from healthy humanvolunteers (males and females between the ages of 22 and 50). Humanumbilical cord blood samples (20-70 ml) from healthy newborns (38-40weeks gestational age, males and females) were collected in sterilesyringes containing citrate phosphate dextrose solution as theanticoagulant. Written informed consent was obtained from all mothersbefore labor and delivery. The Institutional Review Board at the IndianaUniversity School of Medicine approved all protocols.

Buffy Coat Cell Preparation

Human mononuclear cells (MNCs) were obtained from either adultperipheral or umbilical cord blood. Briefly, 20-100 ml of fresh bloodwas diluted one to one with Hanks Balanced Salt Solution (HBSS)(Invitrogen, Grand Island, N.Y.) and overlayed onto an equivalent volumeof Ficoll-Paque (Amersham Biosciences) a ficoll density gradientmaterial. Cells were centrifuged for 30 minutes at room temperature at1800 rpms (740×g). MNCs were isolated and washed three times with EBM-2medium (Cambrex, Walkersville, Md.) supplemented with 10-20% fetalbovine serum (Hyclone, Logan, Utah), 2% penicillin/streptomyocin(Invitrogen) and 0.25 μg/ml of amphotericin B (Invitrogen) (completeEGM-2 medium).

Culture and Quantitative Analysis of Endothelial Outgrowth Cells

Buffy coat MNCs were initially re-suspended in 12 ml of EGM-2 medium(Cambrex) supplemented with 10% fetal bovine serum, 2%penicillin/streptomyocin and 0.25 μg/ml of amphotericin B (completeEGM-2 medium). Four milliliters of cells were then seeded onto threeseparate wells of a six well tissue culture plate (BD Biosciences,Bedford Mass.) previously coated with extra cellular matrix proteinse.g. type I rat tail collagen (BD Biosciences) vitronectin, fibronectin,collagen type 10, polylysine. The plate was incubated at 37° C., 5% CO₂in a humidified incubator. After 24 hours of culture, the non-adherentcells and debris were carefully aspirated, and the remaining adherentcells were washed one time with 2 ml of EGM-2 medium. After washing, 4ml of EGM-2 medium was added to each well. EGM-2 medium was changeddaily until day 7 of culture and then every other day until the firstpassage.

Colonies of cells initially appeared between 5 days and 22 days ofculture and were identified as well circumscribed monolayers ofcobblestone appearing cells (FIG. 1C). Colonies were enumerated byvisual inspection using an inverted microscope at 40× magnification.

For passaging, cells were removed from the original collagen coatedtissue culture plates using 0.05% trypsin-0.53 mM EDTA (Invitrogen),resuspended in 10 ml of EGM-2 media and plated onto 75 cm² tissueculture flasks coated with type I rat tail collagen. Monolayers ofendothelial cells were subsequently passaged after becoming 90-100%confluent.

Culture of HUVECs and HAECs

Two approaches were used to directly isolate the endothelial cells fromarterial or venous vessels. In the first approach, a 20 G blunt endneedle was inserted into one end of an incised vessel and the vascularcontents (plasma with blood cells) were flushed out the opposite endusing sterile saline. Vascular clamps were then applied to isolate eachend of the vessel (3-5 cm in length). A solution of 0.1% collagenase inHanks balanced salt solution (HBSS) was injected through the vessel wallvia a 23 G needle, and the vessel segments were incubated for 5 min at37° C. The vascular clamp from one end of the vessel was then removedand the endothelial cells were expelled via infusion of a celldissociation buffer (Gibco) (injected through the distal end of thevessel opposite the “open” end of the vessel). The vessel segments wereinfused with a minimum of 10 mL of cell dissociation buffer. Thesuspended cells were centrifuged at 350×g and washed in EBM-2 media with10% FBS, counted, and viability checked using Trypan blue exclusion.

The second approach is best suited for large diameter vessels (>1 cm).The vessel was incised along the entire length and opened with theendothelial lumen exposed. Any remaining blood cells and plasma werewashed away with HBSS. The endothelium was removed by firm scraping witha rubber policeman in a single end-to-end motion. The cells adhering tothe rubber policemen were washed free by swirling the policemen in asolution of EBM-2 with 10% FBS in a 6 cm tissue culture well (precoatedwith extracellular matrix proteins). Cells were cultured with visualexamination each day. Colonies of endothelium emerge in 3-10 days. Theadherent endothelial colonies were removed by trypsin-EDTA andtransferred to T 25 flasks that were coated with extracellular matrixproteins. Cryopreserved human umbilical vein endothelial cells (HUVECs)and human aortic endothelial cells (HAECs) were obtained from Cambrex atpassage three. Cells were seeded in 75 cm² tissue culture flasksprecoated with type I rat tail collagen in complete EGM-2 medium forpassage.

Growth Kinetics and Estimate of Replicative Capacity of EPCs.

At the time of first passage cells were enumerated by a trypan blueexclusion assay (Sigma, St. Louis, Mo.). Monolayers of cells were thengrown to 90% confluence and passaged. At each passage, cells wereenumerated for calculation of a growth kinetic curve, populationdoubling times (PDTs), and cumulative population doubling levels(CPDLs).

The number of population doublings (PDs) occurring between passages wascalculated according to the equation: PD=log₂ (C_(H)/C_(S)) where C_(H)is the number of viable cells at harvest and C_(S) is the number ofcells seeded. The sum of all previous PDs determined the CPDL at eachpassage. The PDT was derived using the time interval between cellseeding and harvest divided by the number of PDs for that passage.

MATRIGEL® Assays and Uptake of Acetylated-Low Density Lipoprotein(Ac-LDL or Dil-Ac-LDL)

MATRIGEL® (extracellular matrix proteins) assays were performed.Briefly, early passage (2-3) HPP-ECFC-derived or EPC-derived endothelialcells were seeded onto 96 well tissue culture plates previously coatedwith 30 μl of MATRIGEL® (extracellular matrix proteins) (BD Biosciences)at a cell density of 5000-20,000 cells per well. Cells were observedevery two hours for capillary-like tube formation.

To assess the ability of attached HPP-ECFC and progeny or EPC andprogeny to incorporate Ac-LDL or Dil-Ac-LDL), 10 μg/ml of Ac-LDL(Biomedical Technologies Inc., Stoughton, Mass.) was added to the mediaof cells cultured in a 6 well type I rat tail collagen coated tissueculture plate. Cells were incubated for 30 minutes or 4 hours at 37° C.and then washed three times with phosphate buffered saline (PBS) stainedwith 1.5 μg/ml of DAPI (Sigma) and examined for uptake of Ac-LDL orDil-Ac-LDL by using a fluorescent microscope.

Immunophenotyping of Endothelial Cells by Fluorescence Cytometry

Early passage (1-2) or (3-4) HPP-ECFC and progeny or EPC and progeny(5×10⁵) were incubated at 4° C. for 30-60 minutes with varyingconcentrations of the primary or isotype control antibody as outlinedbelow in 100 μl of PBS and 2% FBS. Cells were washed three times withPBS containing 2% FBS and analyzed by fluorescence activated cellsorting (FACS©) (Becton Dickinson, San Diego, Calif.). Directlyconjugated primary murine monoclonal antibodies against human CD31conjugated to fluorescein isothiocyanate (FITC) (BD Pharmingen, SanDiego, Calif.) were used at a 1:20 dilution, human CD34 conjugated toallophycocyanin (APC) (BD Pharmingen) at a 1:25 dilution, human CD14conjugated to FITC (BD Pharmingen) at a 1:10 dilution, human CD45conjugated to FITC (BD Pharmingen) at a 1:10 dilution, human CD 117conjugated to APC (BD Pharmingen) at a 1:100 dilution, human CD 146conjugated to phycoerythrin (PE) (BD Pharmingen) at a 1:10 dilution,human AC133 conjugated to PE (Miltenyi Biotec, Auburn, Calif.) at a 1:5dilution, human CD141 conjugated to FITC (Cymbus Biotechnology,Chandlers Ford, UK) at a 1:10 dilution, human CD 105 (BD Pharmingen)conjugated to Alexa Fluor 647 (Alexa Fluor 647 monoclonal antibodylabeling kit, Molecular Probes, Eugene, Oreg.) at a 1:100 dilution, andhuman CD 144 conjugated to Alexa Fluor 647 at a 1:100 dilution.

To test for cell surface expression of vascular cell adhesion molecule(VCAM-1) after activation by a cell agonist, serum starved endothelialcells were stimulated with either 10 ng/ml of recombinant humaninterleukin one (IL-1) (Peprotech, Rocky Hill, N.J.) or 10 ng/ml ofrecombinant human tumor necrosis factor-alpha (TNF-α) (Peprotech) for 4hours at 37° C. Following stimulation, cell surface expression of VCAM-1was tested utilizing a primary antibody against human VCAM-1 conjugatedto FITC (BD Pharmingen) at a 1:20 dilution. For all isotype controls forimmunopherotyping and UCAM-1 expression, the following antibodies wereused: mouse IgG_(2a), κ, conjugated to FITC (BD Pharmingen), mouse IgG₁,κ conjugated to FITC (BD Pharmingen), mouse IgG₁, κ conjugated to PE (BDPharmingen), and mouse IgG₁, κ conjugated to APC (BD Pharmingen).

For detection of cell surface expression of von Willebrand factor (vWF)and flk-1, cells were fixed in acetone for 10 minutes at roomtemperature, washed two times with PBS, and blocked and permeabilizedfor 30 minutes with PBS, 3% nonfat dry milk, and 0.1% Triton X-100(Sigma). We used 2 μg/ml of a primary antibody directed against humanvWF (Dako, Carpenteria, Calif.) and a biotinylated primary antibodydirected against human flk-1 (Sigma) at a 1:20 dilution. The secondaryantibody used for vWF was a goat anti-rabbit antibody conjugated to FITC(BD Pharmingen) at a 1:100 dilution and the secondary antibody used forflk-1 was strepavidin conjugated to APC (BD Pharmingen) at a 1:100dilution. For the isotype control for vWF, we used rabbit Ig primaryantibody (Dako) at a 1:100 dilution with anti-rabbit Ig secondaryantibody conjugated to FITC (BD Pharmingen) at a 1:100 dilution. For theisotype control for flk-1, we used a biotinylated mouse IgG₁, κ (BDPharmingen) primary antibody at a 1:100 dilution with a strepavidin APCsecondary antibody (BD Pharmingen) at a 1:100 dilution.

Telomerase Activity Assay

For detection of telomerase activity, the telomeric repeat amplificationprotocol (TRAP) was employed in the form of a TRAP-eze telomerasedetection kit (Oncor, Gaithersburg, Md.). Briefly, 1000 culturedHPP-ECFC or EPC colonies were absorbed onto filter papers and lysed inTRAP assay buffer. The lysed material was subjected to PCR amplificationand the PCR products (6-bp incremental ladder) were electrophoresed on anon-denaturing polyacrylamide gel and visualized by DNA staining orradiolabeled with ³²P. PCR products were loaded as neat or 1/10 or 1/100dilutions and the level of intensity of staining compared to the HELAcell line (1000 cells) positive control.

Thymidine Incorporation Assays

Endothelial colony-derived endothelial cells were deprived of growthfactors and cultured in EBM-2 media supplemented with 5% FBS for 24hours. Next, 3×10⁴ cells were plated in each well of 6-well tissueculture dishes pre-coated with type I collagen and cultured for 16 hoursin EBM-2 media supplemented with 1% FBS. Cells were then cultured inEBM-2 without serum for an additional eight hours to ensure quiescence.Cells were stimulated in EBM-2 media supplemented with 10% FBS with 25ng/ml of recombinant human vascular endothelial growth factor (rhVEGF)(Peprotech), 25 ng/ml of recombinant human basic fibroblast growthfactor (rhbFGF) (Peprotech) or no growth factors, as indicated, in a 37°C., 5% CO₂, humidified incubator. Some cells were cultured in EBM-2media without growth factors or FBS. Cells were cultured for 16 hours,and 1 μCi of tritiated thymidine (Perkin Elmer Life Sciences Products,Boston, Mass.) was added 5 hours prior to the harvest. Cells were lysedwith 0.1 N sodium hydroxide for one hour. Lysates were collected into 5ml of liquid scintilant (Fisher Scientific, St. Louis, Mo.) and βemission was measured. Assays were performed in triplicate.

Generation of GALV-Pseudotyped MFG-EGFP

The MFG-EGFP retrovirus vector expresses the enhanced green fluorescentprotein (EGFP) under the control of the Moloney murine leukemia viruslong terminal repeat (LTR) and has been previously described by Polloket al. (2001). For generation of the GALV-pseudotyped vector,supernatant from an amphotrophic MFG-EGFP clone was used to infect thePG 13 packaging line (American Type Culture Collection (ATCC), Manassas,Va.), and infected cells were isolated by single cell cloning.Individual clones were screened for titer by infecting 5×10⁵ humanerythroleukemic cells (HEL) (ATCC) and determining the percent EGFPexpression 48 hours after end-point dilution of supernatant. MFG-EGFPclone 5 has a titer of 0.5-1×10⁶ infectious units/ml and was used forexperiments.

Retroviral Transduction of Endothelial Cells

Early passage (1-2) endothelial colony-derived endothelial cells weretransduced with equivalent starting titers of MGF-EGFP supernatant. Sixwell non-tissue culture plates were coated with 5 μg/cm² fibronectinCH-296 (Takara Shuzo, Otsu, Japan) for 2 hours at room temperature orovernight at 4° C. Plates were washed one time with PBS, and endothelialcells were plated at 5×10⁴ cells/cm² for transduction. Cells wereinfected with retrovirus supernatant diluted 1:1 with complete EGM-2 for4 hours on 2 consecutive days with a change of complete EGM-2 media forovernight incubation. After the second round of infection, cells wereharvested, counted and analyzed for EGFP expression by fluorescencecytometry.

Single Cell Assays

Early passage (1-4) endothelial colony-derived endothelial cells,transduced with the MFG-EGFP retrovirus, were sorted by fluorescencecytometry for EGFP expression. A FACS Vantage Sorter (Becton Dickenson)was used (sort nozzle ≥100 microns at a sheath pressure of ≤9 pounds persquare inch) to place one single endothelial cell expressing EGFP intoeach well of a 96 well flat bottom tissue culture plate pre-coated withtype I collagen containing 20011 of complete EGM-2 media. Individualwells were examined under a fluorescence microscope at 50× magnificationto ensure that only one cell had been placed into each well. Cells werecultured at 37° C., 5% CO₂ in a humidified incubator. Media was changedevery four days by removing 100 μl and replacing it with 100 μl of freshcomplete EGM-2 media. At day 14, each well was examined for the growthof endothelial cells from the single plated cell. To quantitate thefrequency of dividing single endothelial cells, the number of wells,which had 2 or more endothelial cells with a fluorescent microscope at100× magnification were counted. To enumerate the number of cells perwell, the cells were counted by visual inspection with a fluorescentmicroscope at 100× magnification (less than 50 cells per well), or thecells were trypsinized and counted them with a hemacytometer utilizing atrypan blue exclusion assay (more than 50 cells per well).

The long term proliferative and replating potential of endothelial cellsderived from a single cell was determined. At day 14 after initiation ofculture, individual wells containing greater than 50 cells weretrypsinized, collected in 500 μl of complete EGM-2 media and subculturedto a 24 well tissue culture dish coated with type I collagen. Four daysafter subculturing the cells, the media was aspirated and replaced with500 μl of fresh complete EGM-2 media. On day 7, wells were examined forcolony growth or cell confluence by visual inspection with a fluorescentmicroscope at 50× magnification. Cells were then trypsinized, counted,and subcultured in a 6 well tissue culture plate precoated with type Icollagen. Following 7 days of culture in a six well plate, 10-12 wells,which contained confluent cell monolayers, for long-term cultures wereselected under the conditions disclosed herein. For each sample, PDT andCPDL were calculated.

CD Markers

CD14 (lipopolysaccaride receptor)

CD31 (platelet endothelial cell adhesion molecule)

CD34 (sialomucin)

CD45 (common leukocyte antigen)

CD105 (endoglin)

CD 117 (c-Kit receptor)

CD133 (prominin 1)

CD141 (thrombomodulin)

CD144 (vascular endothelial cadherin)

CD146 (endothelial associated antigen, S-endo-1)

flk-1 (fetal liver kinase-1, receptor for vascular endothelial growthfactor 2)

Confocal Imaging of EPC

Passage 3-5 EPC were grown in a T 75 flask for four days using EBM-2media with 10% added FBS. When cells reached confluence, media wasaspirated, 5 ml of sterile PBS was added to the flask, and thenaspirated, trypsin—EDTA was added and the flask was incubated for 5 minat 37° C. To quench the trypsin, 5 mL of EBM-2 media with 10% FBS wasadded and the released EPC were centrifuged at 350×G for 10 min. Thepelleted cells were washed with PBS and then resuspended in EBM-2 mediawith 10% FBS.

Glass chamber slides (4 chamber configuration; Corning) were coated withextracellular matrix proteins (e.g. collagen type 1 or 4, fibronectin,or vitronectin) over night at 4° C. and then washed with sterile PBS inthe morning. The PBS was aspirated and cells in EBM-2 media with 10% FBSwere added at 50 cells per chamber and incubated at 37° C. in 5% CO₂ for7 days.

EPC containing slides were washed twice with PBS and cells were fixed inacetone for 10 min at room temperature, washed twice with PBS, andblocked and permeabilized for 30 minutes with PBS, 3% nonfat dry milk,and 0.1% Triton X100. To highlight the plasma membrane of the cells, aprimary antibody to CD 146 conjugated to phycoerythrin (PE) was added(1.quadrature.g/mL) to the fixed cells along with 1.5 mg/mL DAPI fornuclear staining. After a 30 minute incubation, cells were washed twicein PBS and examined for fluorescence using a Zeiss 510 confocalmicroscope. An ultraviolet laser (351/364 nm excitation) and ahelium-neon laser (543 nm excitation) were used to excite the DAPI andPE-labeled cells through a 40× water objective with the zoom kept on0.7× magnification. Images were captured in a single plane and displayedas monochromatic images for presentation. NIH Image software was used toquantify the nuclear and cytoplasmic diameters of cells from various EPCcolony types.

TABLE 1 Enumeration of the number and time of appearance of endothelialprogenitor cell colonies isolated from adult peripheral and umbilicalcord blood mononuclear cells. Adult peripheral blood Umbilical CordBlood Number of Day of Number of Day of Colonies/20 ml First Colonies/20ml First Donor Blood Colony Donor Blood Colony 1 0.50 15 1 8.18 7 2 0.8317 2 5.45 6 3 0.35 13 3 13.97 7 4 0.83 21 4 4.92 7 5 0.58 13 5 9.79 7 60.17 22 6 1.14 8 7 0.00 — 7 4.09 10 8 0.60 14 8 9.55 6 9 1.00 18 9 11.336 10 0.40 17 10 0.67 6 11 0.60 14 11 16.00 5 12 0.60 17 12 6.00 7 130.00 — 13 16.00 6 14 0.33 12 15 1.33 11 16 0.67 13 17 0.00 — 18 1.00 16Co-Culture of HPP-ECFC and CD34+ Cells Expands NOD-SCID) RepopulatingCells.

Human CD34+ bone marrow cells, which have previously been shown toharbor marrow repopulating cells in NOD/SCID mice (SRCs) were isolated.Typically 0.5-1.0×10⁶ human marrow CD34+ cells are injected intoNOD/SCID mice in order to achieve a level of human CD45+ chimerism of5-50%. Initially only 9×10³ CD34+ cells were injected into NOD-SCID miceas a control on the day of harvest from human bone marrow. 9×10³ CD34+cells were cultured in the presence of SCF, G-CSF, TPO, and Flt-3 forseven days. These are the growth factors currently used to maximallyexpand HSCs ex vivo. 9×10³ CD34+ cells were co-cultured with monolayersof cord blood HPP-ECFC derived progeny in the absence of growth factorsfor seven days. Following seven days of culture, the cultured CD34+cells were injected into NOD-SCID mice and the peripheral blood oftransplanted mice was tested for the presence of human cells four weeksafter transplantation. Co-culture of CD34+ cells with growth factors for7 days increased the percentage of human cells detected in NOD-SCID mice8 weeks after transplantation 10 fold compared to CD34+ cells injectedshortly after isolation from human bone marrow (FIG. 12A). Despiteinjecting a very limited number of cells compared to prior studies,co-culture of CD34+ cells with cord blood HPP-ECFC-derived cellsincreased the percentage of human cells detected in NOD-SCID mice 8weeks after transplantation 260 fold (FIG. 12C). Both human myeloid andlymphoid lineages were detected eight weeks after transplantationindicating that multilineage reconstitution of the hematopoietic systemwas achieved with CD34+ cells co-cultured with cord blood HPP-ECFC.

Starting with 2 T75 flasks of confluent monolayers of HPP-ECFC-derivedcells, cells were first washed with Hanks balanced salt solution withoutcalcium or magnesium (HBBS), then 1.5 mL of Trypsin EDTA (Gibco) wasadded to each flask for 1 minute. Next 8.5 mL of endothelial basalmedium 2 (EBM2) (Cambrex) with 10% fetal bovine serum (FBS) (Hyclone),was added and suspended cells were collected and counted via Trypan blueexclusion on a hemacytometer.

HPP-ECFC-derived cells were plated at 3×10⁵ cells/well onto collagen 1precoated 6 well tissue culture plates (BD Biosciences). Cells werecultured with endothelial growth medium 2 (EGM2) (Cambrex) supplementedwith 10% FBS and cultured overnight. The following morning the confluentcell monolayers were washed with EBM2+10% FBS twice and then co-culturedwith 9,000 CD34⁺CD38^(dim)Lin⁻ (CD4, 8, 11b, 14, 24, 31, 33, andglycophorin A) adult human bone marrow-derived cells collected byfluorescence activated cell sorting resuspended in 4 mL of EBM2+10% FBS+human megakaryocyte growth derived factor (MGDF) (100 ng/mL),granulocyte colony stimulating factor (G-CSF) (100 ng/mL), and stem cellfactor (SCF) (100 ng/mL), and flt-3 ligand (100 ng/mL). Cells werecultured in 37° C. 5% CO₂ humidified incubator for 7 days withoutdisturbance. In some cultures the CD34⁺ cells were co-cultured with theHPP-ECFC in EBM2+10% FBS and no added growth factors.

A 5 mL pipette was used to aspirate the nonadherent cells and media (4mL) at the end of the 7 day co-culture. Wells were washed once with 2 mLphosphate buffered saline (PBS) and the PBS with nonadherent cells addedto the original aspirate. To the same wells, 1 mL of cell dissociationbuffer (Gibco) was added for 4 minutes at room temperature, and then thecell dissociation buffer and loosened cells were titrated in the wellgently before aspiration and adding to the original aspirate. Finally,the HPP-ECFC monolayers were washed one final time with 2 mL of PBS andthis solution with scant cells was added to the original aspirate. Thefinal volume of media and cells was 9 mL.

The cell suspension was centrifuged at 1500 rpm (514×g) at roomtemperature for 10 minutes. The solution was removed and the cell pelletwas dislodged mechanically then resuspended in ½-1 mL of EBM2+10% FBS.Cells were counted in Trypan blue on a hemacytometer. Recovered cellswere plated in progenitor assays or injected intravenously into NOD/SCIDmice.

The method outlined above may be modified to provide a graft for a humantransplant. In this instance, the HPP-ECFC progeny is plated in T75flasks or in a perfusion chamber system to permit large numbers of CD34+hematopoietic stem cells (autologous or allogeneic human cord blood-,mobilized peripheral blood-, or marrow-derived) to be expanded in thepresence of the cord blood HPP-ECFC. Systems are used that will permitthe donor CD34+ cells to be cultured with the HPP-ECFC progeny withoutthe cells directly touching and, thus, the donor CD34+ cells can beexpanded, recovered, and transplanted into the human patient without thedonor cells being “contaminated” with the cord blood HPP-ECFC progeny.

DOCUMENTS

The following documents are incorporated by reference to the extent thatmethods and compositions disclosed are used in practice of the presentinvention.

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We claim:
 1. A culture method for a high proliferativepotential-endothelial colony forming cell (HPP-ECFC), the methodcomprising: (a) providing a blood sample selected from the groupconsisting of umbilical cord blood and blood vessel blood; (b) isolatinga mononuclear cell from the blood sample; (c) seeding the mononuclearcell on a culture substrate, wherein the culture substrate comprises acoating comprising an extracellular matrix molecule (ECM); (d) culturingthe seeded adherent mononuclear cell in a culture medium for about 5 toabout 10 days to form a colony comprising cells; (e) immunophenotypingthe cells from the colony to determine expression of CD31, CD141, CD105,CD146, CD144, vWF, and flk-1, but not CD45 and CD14; and (f) culturingthe cells that express CD31, CD141, CD105, CD144, CD146, vWF, and flk-1,but do not express CD45 and CD14, wherein the cells have a nucleardiameter ranging from 8 microns to 10 microns.
 2. The method of claim 1further comprising subculturing the cells that express CD31, CD141,CD105, CD146, CD144, vWF, and flk-1, but not CD45 and CD14 for at least30 population doublings.
 3. The method of claim 1 further comprisingsubculturing a single cell that expresses CD31, CD141, CD105, CD146,CD144, vWF, and flk-1, but not CD45 and CD14 on an ECM coated plate toform a secondary colony comprising at least 2000 cells.
 4. The method ofclaim 3, further comprising subculturing a single cell of the secondarycolony on an ECM coated plate to form a tertiary colony.
 5. The methodof claim 1, wherein the culture medium comprises complete EGM-2 media.6. The method of claim 1, wherein the extracellular matrix molecule istype I collagen.
 7. The method of claim 1 further comprising analyzingthe cells that express CD31, CD141, CD105, CD146, CD144, vWF, and flk-1,but not CD45 and CD14 for ingestion of acetylated-low densitylipoprotein.
 8. The method of claim 1, wherein the number of cellsincreases about 10²⁰ fold over a period of 100 days in culture.
 9. Themethod of claim 1, further comprising transducing the cells that expressCD31, CD141, CD105, CD144, CD146, vWF, and flk-1, but do not expressCD45 and CD14 with a nucleic acid encoding a marker.
 10. The method ofclaim 9, wherein the marker is green fluorescent protein.
 11. The methodof claim 9, further comprising selecting a single cell expressing thenucleic acid encoding the marker.
 12. The method of claim 11, whereinthe single cell expressing the nucleic acid encoding the marker isplated.
 13. The method of claim 12, wherein the single cell expressingthe nucleic acid encoding the marker is plated by fluorescent cytometrysorting.
 14. The method of claim 12, wherein the single cell expressingthe nucleic acid encoding the marker forms a secondary colony comprisingat least 2000 cells.
 15. The method of claim 14, further comprisingsubculturing a single cell of the secondary colony on an ECM coatedplate to form a tertiary colony.